Reconstitution of Bovine A1 Adenosine Receptors and G Proteins in Phospholipid Vesicles:  βγ-Subunit Composition Influences Guanine Nucleotide Exchange and Agonist Binding

We have studied the interactions of purified A1 adenosine receptors and G proteins reconstituted into phospholipid vesicles to investigate how the βγ composition of G protein heterotrimers influences coupling. Recombinant hexahistidine-tagged bovine A1 adenosine receptors were expressed in Sf9 cells and purified to homogeneity by sequential chromatography over heparin−sepharose, xanthine amino congener−agarose, and nickel−nitrilotriacetic acid columns. These receptors were reconstituted with pure recombinant G proteins of defined subunit composition. Receptor−G protein complexes containing αi2 and β1γ2 or β1γ3 and stimulated with the agonist, (R)-phenylisopropyladenosine, exchange guanine nucleotide 2−3 times more rapidly than do complexes containing β1γ1. This difference is not overcome by increasing the concentration of βγ subunits. Receptor−G protein complexes containing β1γ1 also bind less of the agonist, [125I]-iodoaminobenzyladenosine (125I-ABA), than do complexes containing β1γ3. Kinetic experiments show that 125I-ABA dissociates 2-fold more rapidly from receptor−G protein complexes containing β1γ1 than from complexes containing the other βγ subunits. The affinity of the interaction between immobilized Gα i2 subunits and β1γ1 or β1γ2 measured with an optical biosensor in the absence of receptor is similar. Taken together, these data implicate the γ-subunit in influencing the interaction between the A1 adenosine receptor and G proteins.